The present invention relates to a high resolution microscopy for the use in ophthalmology.
In particular, the present invention relates to a method, an apparatus (an ophthalmoscope), a computer implemented method and a computer program product for non-invasive observations of the fundus of an eye (eyeground).
Diseases of the eye fundus (e.g. the age-related macular degeneration) are frequently accompanied with microscopic physiological changes, which cannot be diagnosed extrinsically (non-invasively) with conventional methods. Thus for example, age-related macular degeneration is accompanied with the deposit of fluorescent pigments in the retinal pigment epithelium (RPE).
Investigations on histological preparations by means of electron beam microscopy and high resolution light optical methods with a short working distance have revealed that granula are formed as an intracellular correlate of these pigments, the granula having a size of approximately 1 μm.
Due to the distance between the fundus and the eye-lens, the spatial resolution of conventional non-invasive imaging methods is not sufficient to analyse these fine fluorescent structures directly at the patient. Furthermore, there are many disturbing effects due to auto-fluorescence, diffraction and scattering outside the object plane.
Conventional wide-field fundus cameras suffer from a low contrast. Thus, so-called scanning laser ophthalmoscopes (SLOs), which belong to confocal microscopes, have been proposed to investigate the fundus with high resolution. A scanning laser ophthalmoscope scans the fundus point-by-point by a focused laser beam resulting in an improved contrast compared to a conventional fluorescent fundus camera.
In order to further increase the resolution of scanning laser ophthalmoscopes, adaptive optics may be used to compensate for the disturbing effects of lens aberrations. By means of adaptive optics, the resolution of the scanning laser ophthalmoscopes may be increased from 5-10 μm to 2-3 μm. However, the use of adaptive optics is complex and cost-intensive.
Other approaches to increase the resolution and particularly the contrast of images of the fundus use two-photon excitation. The concept of the two-photon microscopy is based on a fluorescence excitation which requires the energy of two photons, i.e., fluorescence excitation only occurs when two photons are absorbed. In such a system, the intensity of fluorescent light emitted by the specimen depends quadratically on the intensity of the excitation light. Therefore, much more two-photon fluorescence is generated where the illumination beam is tightly focused than where it is more diffuse. Accordingly, excitation is restricted to a small focal volume, resulting in an effective rejection of out-of-focus objects. The observed specimen is scanned by the focused excitation light. Usually, also the detection is realized in focus resulting in a confocal microscopy method with a two-photon excitation. Since the probability of the near-simultaneous absorption of two photons is extremely low, high energy densities of the excitation light (laser power and focusing) are required. Therefore, due to temporary high local thermal exposures an application in connection with humans, particularly in ophthalmoscopy, is problematic. Since the fundamental requirements for light hazard protection (ISO 15004-2:2007) have to be met, only relatively small energy densities are allowed in connection with ophthalmic instruments, which may lead to a low signal-to-noise relation. An expensive laser system and a relatively complex arrangement are further drawbacks of the two-photon technique.
Moreover, another difficulty frequently occurring in the field of ophthalmology is that a healthy eye, which is not fixed, permanently carries out eye movements, even when the eye is kept subjectively still. Such movements are usually considered to be obstructive for imaging the fundus. Particularly the quasi-stochastic micro-saccades, i.e. jerky or abrupt movements of the eye typically in the range of 3 to 50 angular minutes, may imply significant lateral shifts of an observed region when observing this region with a high resolution.